Intracellular Chloride Regulation in AVP+ and VIP+ Neurons of the Suprachiasmatic Nucleus

Abstract

Several reports have described excitatory GABA transmission in the suprachiasmatic nucleus (SCN), the master pacemaker of circadian physiology. However, there is disagreement regarding the prevalence, timing, and neuronal location of excitatory GABA transmission in the SCN. Whether GABA is inhibitory or excitatory depends, in part, on the intracellular concentration of chloride ([Cl^-]_i). Here, using ratiometric Cl^- imaging, we have investigated intracellular chloride regulation in AVP and VIP-expressing SCN neurons and found evidence suggesting that [Cl^-]_i is higher during the day than during the night in both AVP+ and VIP+ neurons. We then investigated the contribution of the cation chloride cotransporters to setting [Cl^-]_i in these SCN neurons and found that the chloride uptake transporter NKCC1 contributes to [Cl^-]_i regulation in SCN neurons, but that the KCCs are the primary regulators of [Cl^-]_i in SCN neurons. Interestingly, we observed that [Cl^-]_i is differentially regulated between AVP+ and VIP+ neurons-a low concentration of the loop diuretic bumetanide had differential effects on AVP+ and VIP+ neurons, while blocking the KCCs with VU0240551 had a larger effect on VIP+ neurons compared to AVP+ neurons.

Figure 1

R_Cl measurement in genetically-identified SCN neurons. Confocal micrographs demonstrating regional expression of Cl-Sensor in the SCN of AVP::Cl-Sensor (A) and VIP::Cl-Sensor (B) mice. Native Cl-Sensor fluorescence is depicted in green, and DAPI stain is shown in blue. Pseudocolored epifluorescent micrographs of an AVP::Cl-Sensor SCN slice exhibiting YFP (C) and CFP (D) emission. (E) Baseline R_Cl was higher during the day (ZT 2 to 8) compared to the night (ZT 12 to 18) for both AVP+ (GEE, p < 0.05) and VIP+ (GEE, p < 0.001) neurons.

Figure 2

GABA_A receptor-mediated Cl⁻ transients in AVP+ and VIP+ neurons. Top row: AVP+ neurons from AVP::Cl-Sensor mice tested during subjective day (left) and subjective night (right) demonstrated an increase of R_Cl following puff application of the GABA_A agonist isoguvacine (gray arrow), indicative of inhibitory Cl⁻ influx. Similarly, VIP+ neurons from VIP::Cl-Sensor mice during both day and night responded to isoguvacine with an increase of R_Cl (bottom row). Each trace represents a R_Cl measurement obtained from a single neuronal soma.

Figure 3

The KCCs regulate [Cl⁻]_i in SCN neurons. (A) Example experiment from an AVP::Cl-Sensor mouse recorded during the night demonstrating the effect of 10 µM of the KCC antagonist VU. VU caused an increase in R_Cl indicative of a rise in [Cl⁻]_i. Each trace represents a R_Cl measurement obtained from a single neuronal soma. (B) Example experiment from a VIP::Cl-Sensor mouse recorded during the day demonstrating the effect of VU. VU caused an increase in R_Cl indicative of a rise in [Cl⁻]_i. (C) Summary data of the average change in R_Cl after VU by neuron type and time of day. VU resulted in an increase in R_Cl in all conditions (GEE, p < 0.005), but had a significantly greater effect in VIP+ neurons compared to AVP+ neurons (GEE, p < 0.05). (D) Summary of changes in R_Cl elicited by VU in a HEPES-buffered solution. VU had a larger effect during the day in VIP+ neurons when compared to VIP+ night (GEE, p < 0.05) and AVP+ day neurons (GEE, p < 0.001). For (C) and (D), the number of slices and total regions of interest (in parentheses) is listed for each condition under the x-axis.

Figure 4

NKCC1 contributes to setting resting [Cl⁻]_i in SCN neurons. (A) Example experiment from an AVP::Cl-Sensor mouse recorded during the night showing the effect of blocking NKCC1 with 10 µM of bumetanide. Bumetanide caused a small increase in R_Cl. Each trace represents a R_Cl measurement obtained from a single neuronal soma. (B) Example experiment from a VIP::Cl-Sensor mouse recorded during the night demonstrating the effect of bumetanide. Bumetanide caused a small decrease in R_Cl. (C) Summary data of the average change in R_Cl after bumetanide by neuron type and time of day. Bumetanide elicited small but statistically significant changes in R_Cl in each condition (GEE, p < 0.005). AVP+ and VIP+ neurons responded differently to bumetanide (GEE, p < 0.001), but there were no day/night differences within neuron types. Bumetanide had a statistically different effect on VIP+ neurons compared to AVP+ neurons. (D) Summary of changes in R_Cl elicited by bumetanide in a HEPES-buffered solution. Bumetanide had a larger effect in AVP+ neurons compared to VIP+ neurons (GEE, p < 0.001). For (C) and (D), the number of slices and total regions of interest (in parentheses) is listed for each condition under the x-axis.

Figure 5

Bumetanide occludes the effect of VU. (A) Example experiment from a VIP::Cl-Sensor mouse recorded during the night in which VU (10 µM) was applied after bumetanide (10 µM). From rest, VU elicits a 0.24 increase in R_Cl on average (B, same data from Fig. 3). However, the effect of VU was occluded in the presence of bumetanide (GEE, p < 0.001), suggesting that NKCC1 mediates the Cl⁻ accumulation elicited by VU. Conversely, the effect of bumetanide in the presence of VU was similar to the effect of bumetanide alone (Fig. C and D), indicating that the KCCs are necessary to mediate Cl⁻ extrusion in these neurons.